Spectroscopy ELDOR

M. Bennati, A. Weber, J. Antonie, D.L. Perlstein, J. Robblee and J. Stubbe, Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl radicals in the R2 subunit of the E. coli ribonucleotide reductase, J. Am. Chem. Soc., 2003, 125, 14988. 

Double-resonance spectroscopy involves the use of two different sources of radiation. In the context of EPR, these usually are a microwave and a radiowave or (less common) a microwave and another microwave. The two combinations were originally called ENDOR (electron nuclear double resonance) and ELDOR (electron electron double resonance), but the development of many variations on this theme has led to a wide spectrum of derived techniques and associated acronyms, such as ESEEM (electron spin echo envelope modulation), which is a pulsed variant of ENDOR, or DEER (double electron electron spin resonance), which is a pulsed variant of ELDOR. The basic principle involves the saturation (partially or wholly) of an EPR absorption and the subsequent transfer of spin energy to a different absorption by means of the second radiation, leading to the detection of the difference signal. The requirement of saturability implies operation at close to liquid helium, or even lower, temperatures, which, combined with long experimentation times, produces a... 

Rapatskiy L, Cox N, Ames W, et al. Detection of the water binding sites of the oxygen-evolving complex of photosystem II using W-band 170 ELDOR-detected NMR spectroscopy. J Am Chem Soc 2011 Epub date August 31, 2012. 

There are many other specialized methods electron-electron double resonance (ELDOR), TRIPLE, HYSCORE (hyperfine sublevel correlation spectroscopy, which is similar to 2D-EPR), electron spin-echo, and so on these methods are not discussed here. 

In an early review, Hyde [272] discussed ENDOR in proteins, including flavo-proteins, copper proteins, hemeproteins, two-iron ferrodoxin and bacteriochloro-phyll. Kevan and Kispert s book [19] is an introductory text on ENDOR (electron nuclear double resonance) and ELDOR (electron electron double resonance) techniques. Poole [11] includes a chapter on double resonance techniques in his text. Schweiger [21] has covered ENDOR of transition metal complexes, including a section on biological applications. Recent reviews of ENDOR spectroscopy of chlorophylls [273], heme and heme proteins [274] and iron sulfur proteins [275] demonstrate how additional detail can be obtained from ENDOR data. 

A very important ELDOR mechanism is diffusion of saturation because of very slow rotational diffusion of nearly immobilized species. This is the basis of saturation transfer spectroscopy [57]. 

When tr is sufficiently long, cw EPR spectra become insensitive to nitroxide motion, and the spectra are indistinguishable from powder patterns. Thus, one must rely on spectroscopic methods beyond cw EPR for the extraction of motional processes. Historically, the motional regime between 1(K) nsec and 1 psec has presented a blind spot to EPR spectroscopy, and reliable methods for extraction of t did not exist. More recently, though, this problem has been solved. The technique combines the methods of pulsed saturation recovery EPR (SR-EPR) and pulsed saturation recovery electron-electron double resonance (SR-ELDOR). Spectroscopic saturation occurs when the applied radiation is sufficiently intense... 

Fig. 8. Two-dimensional exchange spectroscopy (often called 2D-ELDOR) of the spin-labeled 3K-8 peptide with mixing time T = 296 nsec. Both the 2D surface and the contour map are shown. The peaks along the diagonal are related to the absorption spectrum of the spin label. The high-held M,= - line is weak because of experimental dead time artifacts. The cross-peaks, especially those between the outermost hyperfine lines, provide direct evidence of Heisenberg spin exchange. The cross-peak intensity can be used to determine the second-order rate constant for collisions between peptides.

Flaender M, Sicoli G, Fontecave T, Mathis G, Saint-Pierre C, Boulard Y, Gambarelli S, Gasparutto D (2008) Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy. Nucleic Acids Symp Ser 52 147-148. doi 10.1093/nass/nm075... 

Abstract Multi-resonance involves ENDOR, TRIPLE and ELDOR in continuous-wave (CW) and pulsed modes. ENDOR is mainly used to increase the spectral resolution of weak hyperfine couplings (hfc). TRIPLE provides a method to determine the signs of the hfc. The ELDOR method uses two microwave (MW) frequencies to obtain distances between specific spin-labeled sites in pulsed experiments, PELDOR or DEER. The electron-spin-echo (ESE) technique involves radiation with two or more MW pulses. The electron-spin-echo-envelope-modulation (ESEEM) method is particularly used to resolve weak anisotropic hfc in disordered solids. HYSCORE (Hyperfine Sublevel Correlation Spectroscopy) is the most common two-dimensional ESEEM method to measure weak hfc after Fourier transformation of the echo decay signal. The ESEEM and HYSCORE methods are not applicable to liquid samples, in which case the FID (free induction decay) method finds some use. Pulsed ESR is also used to measure magnetic relaxation in a more direct way than with CW ESR. 

There are a variety of techniques for the determination of the various parameters of the spin-Hamiltonian. Often applied are Electron Paramagnetic or Spin Resonance (EPR, ESR), Electron Nuclear Double Resonance (ENDOR), Electron Electron Double Resonance (ELDOR), Nuclear Magnetic Resonance (NMR), occassionally utilizing effects of Chemically Induced Dynamic Nuclear Polarization (CIDNP), Optical Detection of Magnetic Resonance (ODMR), Atomic Beam Spectroscopy and Optical Spectroscopy. The extraction of the magnetic parameters from the spectra obtained by application of these and related techniques follows procedures which may in detail depend on the technique, the state of the sample (gaseous, liquid, unordered solid, ordered solid) and on spectral resolution. For particulars, the reader is referred to the general references (D). 

Not all the information can be obtained by the basic CW experiment that is considered by many chemists as all there is to EPR. Elucidating geometric structure or small spin densities requires the separation of small hyperfine couplings or dipole-dipole couplings between electron spins from larger interactions. This can be achieved by double resonance experiments, such as electron nuclear double resonance (ENDOR) [8,9] and electron electron double resonance (ELDOR) spectroscopy and further pulse-EPR techniques [10] such as electron spin echo envelope modulation (ESEEM). Pulse-EPR techniques may also provide more information on dynamic processes than simple CW experiments and may access longer time scales. 

Figure 1. Scheme of the pulse EPR sequences mentioned in this chapter, (a) Two-pulse ESEEM. (b) Three-pulse ESEEM. (c) Four-pulse ESEEM. When times fi and ti are stepped under the constraint of ti= ti= T, combination-peak experiment is performed. Two-dimensional HYSCORE spectroscopy is done using the same sequence, whereby t and are stepped independently. The second and third nil pulse are replaced by high-tuming-angle (HTA) pulses in a matched HYSCORE experiment, (d) SMART-HYSCORE. The first and third pulses are HTA pulses, (e) Davies ENDOR. (f) Mims ENDOR. (g) ELDOR-detected NMR. 

The size of the spin-spin interaction of the [NiFe] center and the [3Fe4S] cluster (S = Vi) is within the range that can be studied by pulse ELDOR spectroscopy. This PELDOR technique allows measurement of the spin-spin interaction and a determination of the effective distance between the two electron spins. Measurements have so far only been performed for D. vulgaris Miyazaki F hydrogenase [94] on the as-isolated enzyme (30% Ni-A and 70% Ni-B). The spin delocalization over the [3Fe4S] cluster had to be included for correct data analysis. Spin projection coefficients have been determined that indicate that the largest amount of eleetron spin density is located on the iron closest to the [NiFe] center. 

A large variety of hyperfine spectroscopy methods exist that allow the detection of hyperfine and nuclear quadrupole interactions electron spin-echo envelope modulation (ESEEM), ENDOR, and ELDOR-detected NMR (electron-electron doubleresonance detected nuclear magnetic resonance) [13]. Although there are cases in which ESEEM and ENDOR perform equally well, ESEEM-like methods tend to be... 

In polymeric liquid crystals, not only the mobility in the liquid-crystalline phase but also the highly restricted dynamics of the probe in the frozen state can be studied by 2D ELDOR spectroscopy [ 140]. In high performance materials based on main chain liquid crystalline polymers, stable radicals are created during production and processing. The nature and the dynamics of these defects has been elucidated by pulsed electron-nuclear double resonance (ENDOR) spectroscopy [141],... 

---